Transglutaminases form a large family of protein cross-linking enzymes. Six transglutaminase gene products, mentioned below, have been characterized in higher vertebrates on the basis of their primary structure. Aeschlimann, D. and Paulsson, M. (1994) Thromb Haemostasis 71: 402-415. Enzymes of this class catalyze the Ca.sup.2+ -dependent transferase reaction (EC 2.3.2.13) which leads to the formation of an isopeptide bond between the .gamma.-carboxamide group of a peptide-bound glutamine residue and various primary amines. Folk, J. E., and Finlayson, J. S. (1977) Adv. Protein Chem. 31:1-133; Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 58:9-35. Most commonly, .gamma.-glutamyl-.epsilon.-lysine cross-links are formed in or between proteins by reaction with the .epsilon.-amino group of lysine residues. Analysis of the three-dimensional structure of the a-subunit of factor XIII showed that transglutaminases contain a central core domain containing enzymatic activity, and a N-terminal .beta.-sandwich domain and two C-terminal .beta.-barrel domains which are presumably involved in regulation of enzyme activity and specificity. Yee, V. et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7296-7300. The catalytic core domain of transglutaminases is structurally related to the cysteine proteases and forms a similar catalytic triad, Cys-His-Asp, in the enzyme active site. This provides strong evidence for the transglutaminase cross-linking reaction being the reverse of the proteolytic cleavage reaction catalyzed by cysteine proteases. Yee, V. et al. (1996) Sem. Thromb. Haemostasis 22:377-384. Transglutaminases undergo a number of post-translational modifications such as phosphorylation, fatty acylation, and proteolytic cleavage which regulate their enzymatic activity and sub-cellular localization. Aeschlimann, D. and Paulsson, M. (1994) Thromb. Haemostasis 71:402-415.
Transglutaminase C ("TG.sub.C "; tissue transglutaminase, transglutaminase type II) is expressed in many cell types and tissues in the vertebrate body, including endothelial cells, fibroblasts, macrophages, erythrocytes, chondrocytes, hepatocytes, smooth muscle cells, astrocytes, heart muscle, spleen, lung, eye lens, and various epithelia such as intestinal epithelia, tracheal epithelia, mucosal epithelia, mammary epithelia, kidney tubular epithelia, etc. Thomazy, V. and Fesus, L. (1989) Cell Tissue Res. 255: 215-224; Aeschlimann, D. and Paulsson, M. (1991) J. Biol. Chem. 266:15308-15317; Gentile, V. et al., (1991) J. Biol. Chem. 266:478-483; Weraarchakul-Boonmark, N. et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89: 9804-9808; Aeschlimann, D. et al. (1993) J. Cell Biol. 120: 1461-1470. In contrast to the other transglutaminase family members, the physiological function of TG.sub.C remains unclear and might be diverse in different tissues or biological events. TG.sub.C has been implicated in diverse processes such as stabilization of extracellular matrices in development and in wound healing, in apoptosis, and in receptor signaling. Aeschlimann, D. and Paulsson, M. (1991) J. Biol. Chem. 266: 15308-15317; Fesus, L. et al. (1991) Eur. J. Cell Biol. 56: 170-177; Aeschlimann, D. et al. (1995) J. Cell Biol. 129: 881-892; Nakaoka, H. et al. (1994) Science 264: 1593-1596.
Band 4.2 protein ("Band 4.2") is a membrane cytoskeleton component expressed at high level in erythroid cells. Korsgren, C. et al. (1990) Biochemistry 87, 613-617; Risinger, M. D, et al. (1992) J. Biol. Chem. 267, 5680-5685. Band 4.2 protein is the only member of this gene family that has lost the enzymatic activity to become a purely structural protein.
Platelets are the major source for factor XIII a-subunit ("FXIIIa") in plasma. Grundmann, U. et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8024-8028; Takahashi, N. et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8019-8023; Poon, M. et al. (1989) J. Clin Invest 84, 787-89. Platelets have been shown to contain mRNA transcripts even though they lack a nucleus, e.g. for FXIIIa (see below). Sottile, J. et al. (1989) Thrombosis Haemost. 62, 1100-1102. FXIIIa stabilizes the fibrin clot in haemostasis. Congenital deficiencies or acquired autoimmune response to factor XIII lead to a delayed bleeding tendency even though the primary haemostasis is normal due to insufficient clot stability. Board, P. G. et al. (1993) Blood Rev. 7: 229-242.
Transglutaminase K ("TG.sub.K "; keratinocyte transglutaminase; transglutaminase type I) and transglutaminase E ("TG.sub.E "; epidermal transglutaminase, transglutaminase type III) contribute to the formation of the cornified envelope in skin in distinct steps of keratinocyte differentiation. Kim, H. C. et al. (1991) J. Biol. Chem. 266: 536-539; Kim, I. G. et al. (1993) J. Biol. Chem. 268: 12682-12690; Kim, S. Y. et al. (1995) J. Biol. Chem. 270: 18026-18035. A congenital keratinization disorder, a distinct form of the heterogenous group of skin diseases referred to as autosomal recessive lamellar ichthyosis, has been linked to mutations in the gene coding for keratinocyte transglutaminase. Huber, M. et al. (1995) Science 267, 525-528.
Transglutaminase P ("TG.sub.P "; prostate transglutaminase, transglutaminase type IV) is an androgen regulated protein involved in semen coagulation and its expression is restricted to prostate. Ho, D. C. et al. (1992) J. Biol. Chem. 267:12660-12667; Grant, F. J. et al. (1994) Biochem. Biophys. Res. Commun. 203: 1117-1123; Dubbink, H. J. et al. (1996) Biochem. J. 315: 901-908.
A phylogenetic analysis of the transglutaminase genes indicates an early gene duplication event which subsequently gave rise to two different lineages; one including TG.sub.C, TG.sub.E, and Band 4.2 protein; the other, FXIIIa, TG.sub.K, and likely also TG.sub.P (Aeschlimann and Paulsson, 1994).
Because more than one type of transglutaminase can be expressed in a single cell type (e.g., keratinocytes), and since the same gene product can be present in different cellular compartments, conflicting results have been reported about the nature of the transglutaminase enzymes involved in particular biological processes. What is desired is a sensitive and rapid diagnostic assay to determine the transglutaminases involved in particular biological events. Antibodies are not well suited for distinguishing among transglutaminases because of the potential cross reactivity among the different enzymes and the limited reactivity across species.
Extensive efforts have also been made to develop applications based on the unique ability of transglutaminases to cross-link proteins. Microbial transglutaminases have found use in food processing to add texture to processed foods, in particular processed meat. Food Research and Development Laboratories, Ajinomoto Co., Inc., Kanagawa, Japan. FXIIIa and more recently TG.sub.C have found applications as biological glues. Schlag, G., and Redl, H. (1988) Clin. Orthop. 227: 269-285; Martinowitz, U., and Schulman, S. (1995) Thromb. Haemostasis 74:486-492; Juergensen, K. et al. (1997) J. Bone Joint Surg. 79-A:185-193. Initially, FXIIIa was used as a cryoprecipitate from plasma, a product which carries an inherent risk of pathogen contamination. This has been overcome with the availability of recombinant FXIIIa. FXIIIa is also used therapeutically in patients deficient in factor XIII in the form of repeated intravenous injections. More recently, recombinant FXIIIa was also successfully used to treat chronic wound conditions such as ulcerative leg disease. Wozniak, G. et al. (1996), Sem. Thromb. Haemostasis 22: 445-450. Gene therapy with autologous bone marrow-derived stem cells transfected with an intact copy of the factor XIII gene might become an option for patients with a congenital deficiency of factor XIII. Similarly, the severe skin condition associated with TG.sub.K deficiency might be treated by gene therapy although the technology for the latter tissue is not as far developed. It is likely that the list of pathologies associated with deficiencies of transglutaminases will grow as additional information on the gene level becomes available. Thus, novel genetic therapies that employ recombinant versions of transglutaminase genes are also sought.